A labile selenium donor compound, selenophosphate, is formed from selenide and ATP by selenophosphate synthetase. A divalent metal ion, Mg2+, and a monovalent cation, K+, NH4+ or Rb+, are required for selenophosphate synthetase activity. Na+ and Li+ are ineffective as activators and in the presence of K+ are inhibitory. Mn-ATP, although not able to replace Mg-ATP for catalytic activity, binds to the enzyme provided an active monovalent cation is present. No Mn-ATP is bound when K+ is replaced with Na+. The requirement for K+, both for Mn-ATP binding and for catalytic activity of the synthetase, indicates a specific monovalent cation induced conformational state of the enzyme. Activity of the enzyme is markedly inhibited by fM levels of Zn2+ in the presence of mM levels of Mg2+. Binding of Mn-ATP also is decreased upon addition of Zn2+ indicating that the inhibitory effect of zinc is exerted at the substrate binding step of the overall selenophosphate synthetase reaction. When a cysteine residue at position 17 or 19 is replaced with serine, Mn-ATP binding to these mutant enzymes is unaffected by Zn2+ addition. Direct involvement of these cysteine residues in the zinc binding site was shown using 65ZnCl2. Radioactive zinc bound to wild type enzyme and was retained after gel filtration but under the same conditions the catalytically inactive Cys-17 mutant protein and the catalytically active Cys-19 mutant enzyme were unlabeled.